Biological Optimization Systems For Enhancing Photosynthetic Efficiency And Methods Of Use

ABSTRACT

The present disclosure relates to biological optimization systems for enhancing photosynthetic efficiency and methods of use.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims priority to co-pending U.S. provisionalapplication entitled “Biological Optimization Systems for EnhancingPhotosynthetic Efficiency and Methods of Use,” having Ser. No.61/298,248 filed on Jan. 26, 2010, which is entirely incorporated hereinby reference.

BACKGROUND

It has long been known that the “Flashing Light Effect” inPhotosynthesis can enhance the light utilization efficiency leading tobetter productivity. The goal is to apply a photon flux density that isjust enough to excite the majority of the light harvesting complexes toattain the maximum rate of growth, while simultaneously minimizingtrapped surplus light, which renders losses in the form of heat andfluorescence. The excess absorbed light energy can cause damage to thephotosynthetic apparatus from the reactive free oxygen radicalsgenerated, known as photoinhibition. Thus, by using intermittent light,the number of excitations arriving at a closed reaction center decreaseswhen flashes are shortened, permitting more efficient usage of light andless photodamage repair. The major potential boosts in bioproductivitystems from improving flux tolerance rather than from augmentingintrinsic photosynthesis efficiency. The ultimate rate limiting processfor improving photonic flux tolerance and thus bioproductivity is thetime scale for the dark reactions in algal photosynthesis. The matchingof pulse duration, color spectrum, and instantaneous light intensity ofthe LED light output to the chlorophyll absorption, and subsequent darkreaction kinetics are the key to realizing superior flux tolerance.

Photosynthetic organisms use various pigments to absorb and convertlight energy into chemical energy through photosynthesis. These pigmentshave specific wavelengths of light that are most strongly absorbed, withchlorophyll being the dominant and most important pigment forphotosynthesis. Using colors of light that match the absorption ofpigments in a particular organism has been shown to be more effectivefor driving photosynthesis over using full spectrum or weakly absorbedcolors. However, as pigment densities increase, such as in the case ofhigh density algal cultures, strongly absorbed wavelengths of light,such as blue and red, become very strongly absorbed at the surface ofthe culture and less light is allowed to penetrate deep into theculture. The most popular physical observable used to assessphotosynthetic function and its subsequent down regulation inexcess-light conditions is chlorophyll (Chl) fluorescence, because it issensitive to a wide range of changes in the overall apparatus. Despitedecades of research on the flashing light effect, there have not beenany studies on the apparent increase in photon utilization efficiency(yield) or a minimization of non-photochemical quenching (NPQ), or heatdissipation using PAM Fluorometry. Thus, a need exists to address thesedeficiencies.

SUMMARY

Embodiments of the present disclosure, in one aspect, relate tobiological optimization systems for enhancing photosynthetic efficiencyand methods of use.

Briefly described, embodiments of the present disclosure include methodsfor enhancing photosynthetic efficiency, among others, including:applying pulsed light to a photosynthetic organism; using a chlorophyllfluorescence feedback control system to determine one or morephotosynthetic efficiency parameters, wherein the photosyntheticefficiency parameters are used to adjust one or more of the following: apulse rate, pulse on/off duration, light intensity, light spectrum, or acombination thereof; and adjusting one or more of the photosyntheticefficiency parameters to drive the photosynthesis by the delivery of anamount of light to optimize light absorption of the photosyntheticorganism while providing enough dark time between light pulses toprevent oversaturation of the chlorophyll reaction centers.

Briefly described, embodiments of the present disclosure include abiological optimization system for enhancing photosynthetic efficiencyof a photosynthetic organism including a source of pulsed light and achlorophyll fluorometer.

BRIEF DESCRIPTION OF THE DRAWINGS

Many aspects of the disclosure can be better understood with referenceto the following drawings. The components in the drawings are notnecessarily to scale, emphasis instead being placed upon clearlyillustrating the principles of the present disclosure. Moreover, in thedrawings, like reference numerals designate corresponding partsthroughout the several views.

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

FIG. 1 is a graph that illustrates absorbance spectra of freechlorophyll a and b in a solvent. The spectra of chlorophyll moleculesare slightly modified in vivo depending on specific pigment-proteininteractions.

FIG. 2 is a graph that illustrates the absorption maxima of chlorophylla are lambda=430 and lambda=662 nm, that of chlorophyll b are at 453 and642 nm. Royal Blue Luxeon K2 LED emits in the 440 nm to 460 nm rangewith a peak emission at 455 nm, with spectral half-width of 20 nm, veryappropriate to excite chlorophyll a and nearly an exact match to excitechlorophyll b. Red Luxeon K2 LED emits in the 620.5 nm to 645 nm, withpeak emission at 627 nm and spectral half-width of 20 nm, appropriate toexcite chlorophyll b. Therefore, it is NOT obvious, based solely onChlorophyll a and b spectra, that green light would enhance algae growthand metabolism.

FIG. 3 illustrates a model of a Biofeedback System for OptimizingPhotosynthesis using pulse width modulated LEDs and PAM fluorometry.

FIG. 4 is a graph that illustrates Electron Trapping Efficiency vs. PARsaturation.

FIG. 5 illustrates an experimental setup as discussed in Example 1,below.

FIG. 6 is a graph that illustrates radiometric output for PWM-LED's.

FIG. 7 is a graph that illustrates Effective Photosynthetic Efficiency(Yield) of C. sorokiniana.

FIG. 8 is a graph that illustrates Photochemical quenching (qP) of C.sorokiniana.

FIG. 9 is a graph that illustrates Non-Photochemical Quenching (NPQ) ofC. sorokiniana.

FIG. 10 is a graph that illustrates Effective Photosynthetic Efficiency(Yield) of C. minutissima.

FIG. 11 is a graph that illustrates Photochemical Quenching (qP) of C.minutissima.

FIG. 12 is a graph that illustrates Non-Photochemical Quenching (NPQ) ofC. minutissima.

FIG. 13 is a graph that illustrates the theoretical optimal dark periodto minimize NPQ.

FIG. 14A is a graph that illustrates visible light absorbance ofChlorella sorokiniana at three OD's: 0.5 (a), 1.0 (b), and 1.5 (c). FIG.14B is a graph that illustrates visible light absorbance of differentage Chlorella sorokiniana 7 days (a), 15 days (b), and 30 days (c)diluted to the same OD 0.55.

FIG. 15 illustrates results for LEDs φ_(PSII) arranged by the lowest tothe highest at different culture Optical Density (OD). The LEDscirculated by the same arrows do not show significant differences.Factorial Design analyzes were performed within each OD. Results fromdifferent ODs cannot be compared.

DETAILED DESCRIPTION

Before the present disclosure is described in greater detail, it is tobe understood that this disclosure is not limited to particularembodiments described, as such may, of course, vary. It is also to beunderstood that the terminology used herein is for the purpose ofdescribing particular embodiments only, and is not intended to belimiting, since the scope of the present disclosure will be limited onlyby the appended claims.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit (unlessthe context clearly dictates otherwise), between the upper and lowerlimit of that range, and any other stated or intervening value in thatstated range, is encompassed within the disclosure. The upper and lowerlimits of these smaller ranges may independently be included in thesmaller ranges and are also encompassed within the disclosure, subjectto any specifically excluded limit in the stated range. Where the statedrange includes one or both of the limits, ranges excluding either orboth of those included limits are also included in the disclosure.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this disclosure belongs. Although any methods andmaterials similar or equivalent to those described herein can also beused in the practice or testing of the present disclosure, the preferredmethods and materials are now described.

All publications and patents cited in this specification are hereinincorporated by reference as if each individual publication or patentwere specifically and individually indicated to be incorporated byreference and are incorporated herein by reference to disclose anddescribe the methods and/or materials in connection with which thepublications are cited. The citation of any publication is for itsdisclosure prior to the filing date and should not be construed as anadmission that the present disclosure is not entitled to antedate suchpublication by virtue of prior disclosure. Further, the dates ofpublication provided could be different from the actual publicationdates that may need to be independently confirmed.

As will be apparent to those of skill in the art upon reading thisdisclosure, each of the individual embodiments described and illustratedherein has discrete components and features which may be readilyseparated from or combined with the features of any of the other severalembodiments without departing from the scope or spirit of the presentdisclosure. Any recited method can be carried out in the order of eventsrecited or in any other order that is logically possible.

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how toperform the methods and use the compositions and compounds disclosed andclaimed herein. Efforts have been made to ensure accuracy with respectto numbers (e.g., amounts, temperature, etc.), but some errors anddeviations should be accounted for. Unless indicated otherwise, partsare parts by weight, temperature is in ° C., and pressure is at or nearatmospheric. Standard temperature and pressure are defined as 20° C. and1 atmosphere.

It must be noted that, as used in the specification and the appendedclaims, the singular forms “a,” “an,” and “the” include plural referentsunless the context clearly dictates otherwise. Thus, for example,reference to “a support” includes a plurality of supports. In thisspecification and in the claims that follow, reference will be made to anumber of terms that shall be defined to have the following meaningsunless a contrary intention is apparent.

Definitions:

The terms “algae” and “algal cells” as used herein refer to a large anddiverse group of simple, typically autotrophic organisms, ranging fromunicellular to multicellular forms. They are photosynthetic, likeplants, and “simple” because they lack the many distinct organs found inland plants. All true algae have a nucleus enclosed within a membraneand chloroplasts bound in one or more membranes. “Microalgae” or“microphytes” (also referred to as phytoplankton, or planktonic algae)are microscopic algae, typically found in freshwater and marine systems.There are 200,000-800,000 species exist of which about 35,000 speciesare described. They are unicellular species which exist individually, orin chains or groups. Depending on the species, their sizes can rangefrom a few micrometers (μm) to a few hundreds of micrometers. Microalgaeproduce approximately half of the atmospheric oxygen and usesimultaneously the greenhouse gas carbon dioxide to growphotoautotrophically. The biodiversity of microalgae is enormous andthey represent an almost untapped resource. The chemical composition ofmicroalgae is not an intrinsic constant factor but varies over a widerange, both depending on species and on cultivation conditions.Microalgae such as microphytes constitute the basic foodstuff fornumerous aquaculture species, especially filtering bivalves. Theyprovide them with vitamins and polyunsaturated fatty acids, necessaryfor the growth of the bivalves which are unable to synthesize itthemselves.

“Quantum Yield” (Yield) refers to the proportion of light absorbed bychlorophyll associated with PSII that is used in photochemistry.

“Non-photochemical Quenching” (NPQ) refers to thermal dissipation ofabsorbed energy within the PSII pigment antenna and/or RCII.

“Chlorophyll Fluorescence” refers to re-emission of energy in the formof a photon (light) as an electron returns to ground state from asinglet excited state.

“Pulse-Amplitude-Modulation Fluorometry” refers to the measure of thefluorescence characteristics of the different states of a reactioncenter. Using a probe flash PAM measures the changes in the quantumyield of fluorescence.

The “Feedback Control System” refers to a system that is based onfluorescent measurements of fluorescence from the chlorophyll (asmeasured by the fluorometer), where the system is capable of immediatedetection of responses from the photosynthetic organism. A device (e.g.,a datalogger, a computer (hardware and/or software) in communicationwith the Fluorometer, and the LED illumination system (e.g., one or moreLEDs that can be individually controlled by the device and/or manually,where each LED can be emit at a different wavelength(s)), can adjust thelight intensity, spectrum, and/or pulse of one or more of the LEDs tobetter match the needs of photosynthetic organism so to maximize thegrowth of the photosynthetic organism and maximize the energy efficiencyof the LED illumination system.

Discussion:

In accordance with the purpose(s) of the present disclosure, as embodiedand broadly described herein, embodiments of the present disclosure, inone aspect, relate to biological optimization systems for enhancingphotosynthetic efficiency and methods of use.

In high density algal cultures, high agitation and turnover rates can beused to provide more cells a better opportunity to absorb light energyfor photosynthesis. As the density of algae, or concentration ofchlorophyll in a given area increases, the color of actinic light can beadjusted from blue (440-490 nm), which is strongly absorbed, to cyan(505 nm) or green (530 nm), which is more weakly absorbed but has higherinternal reflection allowing the light energy to penetrate deeper intothe culture or canopy. This can allow an overall increase inphotosynthesis in the given area. As photosynthetic organisms age, thedistribution of pigments may change, which can alter the optimalabsorption spectra for a given organism. Measuring the changes inpigment density and optimal color absorption in real-time, allows for anautomated optimization (e.g., adjust wavelength, pulse rate, lightintensity, and the like) of light absorption by a given photosyntheticorganisms culture or canopy.

Chlorophyll fluorescence may be used to assess photosynthetic functionand its subsequent down regulation in excess-light conditions. By usingdata from fluorometry (e.g., PAM fluorometry) on photosynthetic yieldand/or NPQ from chlorophyll fluorescence, and coupling this informationin real-time for feedback control to adjust actinic light parameters fordriving photosynthesis, such as pulse on/off duration, LED color, and/orlight intensity, one can optimize (e.g., increase) photosyntheticefficiency and biomass productivity.

Embodiments of the present disclosure include photosynthetic organismsincluding a plant or animal utilizing chlorophyll as an energycollector/converter. In an embodiment, the photosynthetic organism caninclude microalgae, macroalgae, terrestrial plant, coral, corallimorph,anemone, claim, a host organism containing a photosynthetic symbioticorganism, and a combination thereof. In another embodiment, themicroalgae can include Chlorella sorokiniana, Chlorella minutissima, anda combination thereof.

Embodiments of the present disclosure can include a biologicaloptimization system where the chlorophyll fluorometer provideschlorophyll fluorescence feedback to a chlorophyll fluorescence feedbackcontrol system (e.g., a fluorometer, datalogger or computer system, andan LED illumination system) where the chlorophyll fluorescence feedbackcontrol system adjusts the output of the source(s) of pulsed light. Inan embodiment, the chlorophyll fluorescence feedback includesphotosynthetic efficiency (quantum yield), photochemical processing(qP), and/or waste heat dissipation (NPQ or qN) of the photosystem. Theparameters can be calculated based on chlorophyll a fluorescenceemissions. In an embodiment, the chlorophyll fluorescence feedback canbe utilized to adjust one or more of: a pulse rate, pulse on/offduration, light intensity, and/or light spectrum, of the source ofpulsed light to provide an amount of light to excite the photosyntheticorganism without oversaturation and photoinhibition with less energyloss through heat dissipation (NPQ). In an embodiment of the presentdisclosure, the pulse rate, pulse on/off duration, light intensity,and/or light spectrum are adjusted during illumination based upon thechlorophyll fluorescence feedback data in real time. In an embodiment,the need for non-photochemical quenching by the organism is reduced, ascompared to continuous illumination systems.

Embodiments of the present disclosure include a biological optimizationsystem where the light intensity of the spectral composition of lightfrom the illumination can change by decreasing the intensity ofwavelengths that are strongly absorbed and increasing the intensity ofwavelengths that are weakly absorbed to allow deeper penetration oflight energy into a culture or canopy of the photosynthetic organism.The change in illumination spectrum is based upon changes in the ratioand quantity of the composition of photosynthetic pigments in thetargeted organism.

In an embodiment, the illumination applies blue (440-490 nm) and/or red(600-680 nm) light to the culture or canopy, when the culture or canopyhas strong absorption in the blue (440-490 nm) and/or red (600-680 nm)regions.

Embodiments of the present disclosure include a biological optimizationsystem where the illuminated culture increases its density duringcultivation preventing effective light penetration into the culture orcanopy and inducing increased energy dissipation as heat (NPQ), wherethe biological optimization system decreases the intensity of blueand/or red illumination and replaces the intensity of illumination withone or more colors having higher reflection, wherein the colors havinghigher reflection can include cyan (495-515 nm), green (520-540 nm),orange/amber (565-595 nm), and a combination thereof. Other types ofadjustments to one or more of the pulse rate, pulse on/off duration,light intensity, or light spectrum, of the source of pulsed light can beadjusted in a similar fashion as the photosynthetic organism grows andchanges.

As mentioned above, embodiments of the present disclosure can include abiological optimization system for enhancing photosynthetic efficiencythat includes a source of pulsed light (e.g., LED illumination system)and a chlorophyll fluorometer (e.g., a Pulse Amplitude Modulation (PAM)fluorometer). In an embodiment, the system enhances photosyntheticefficiency of a photosynthetic organism. Embodiments of the presentdisclosure include a biological optimization system where trappedsurplus light is minimized. In an embodiment, the energy required by thesource of pulsed light is minimized. Additional details are provided inthe Examples.

As mentioned above, embodiments of the present disclosure includeoptimization of photon utilization efficiency (e.g., increase power use)and/or photoinhibition prevention in photosynthetically active organismsunder pulsed actinic illumination. In an embodiment, PAM fluorometry maybe used as a diagnostic tool to measure fluorescence from thephotosynthetic organism. In another embodiment, data acquired using thePAM fluorometer can provide an assessment of the overall photosyntheticstate of the photosynthetic organism. The PAM fluorometer providesinformation (e.g., photosynthetic efficiency parameters) about aphotosynthetic organism's use of chlorophyll for absorbing light energy.The PAM can be used diagnostically to determine what fraction ofabsorbed light is used for photochemistry, re-emitted as fluorescence,and/or dissipated as heat energy. These three fates of absorbed lightenergy provide an instantaneous assessment of how efficient the organismis operating under its current environmental conditions.

Embodiments of the present disclosure can provide real time informationand fast dynamic tuning of the LED illumination system in an automated,compact package. By combining the fluorometer (e.g., fluorometer PAM)and LED illumination system (source of pulsed light from LED(s) at oneor more wavelengths) and coupling them with a feedback loop that takesinformation from the fluorometer, the data then can be used to adjustthe LED(s) light output (e.g., intensity, color, and/or pulse on/offduration). In an embodiment, the information from the PAM fluorometercan be analyzed using an algorithm. In other words, the LED light fromthe LED illumination system can become a “smart” grow light, in thesense that it can maximize the efficient absorption and conversion ofeach photon emitted from the LED(s), whereby maximizing thephotosynthetic efficiency as well as maximizing the energy used by theLED illumination system. As the PAM fluorometer measures the chlorophyllfluorescence of an organism with and without LED illumination, theresultant data on the efficiency and waste of its current photosyntheticperformance can be used to adjust and dynamically tune the output energy(e.g., intensity, color, and/or pulse on/off duration) from the LEDillumination in real-time to maximize the performance of the organism.

Embodiments of the present disclosure may be used to optimize artificialillumination (e.g., from the LED illumination system) for cultivation ofplants or algae. In an embodiment, microalgae cultivation for food,feed, nutraceutical, and biofuel production, agriculture, horticulture,and/or coral aquaculture may be realized. In situations where it iseconomical to use artificial lighting for the cultivation of high valuephotosynthetic organisms, embodiments of the present disclosure canpotentially increase the biomass productivity and/or light utilizationefficiency, i.e., the energy used to generate light in thephotosynthetically active region (PAR) can be dramatically decreased bybeing able to maximize the absorption and utilization (throughphotochemical conversion/photosynthesis) of the light energy by theorganism. In an embodiment, use of high efficiency LED's that are beingpulsed with 50% or less on-time at monochromatic colors can optimizeabsorption into the culture or canopy, which can lead to decrease inenergy costs. The reduced on-time of one or more of the LEDs candecrease electricity usage directly and indirectly by reducing thecooling requirements, which is particularly applicable in indoorenclosures or heat sensitive operations.

In general, the artificial lighting currently used in industry forphotosynthetic organisms, such as metal halides, high pressure sodium,mercury-arc, etc, has tremendous losses associated with the generationof PAR light, due to losses in the IR and UV light region, electronicballast inefficiencies, and external chiller/cooling requirements forenclosed operations. In addition, these high intensity lights canreadily induce photoinhibition and/or photodamage that can decreaseproductivities. These lights also do not have the ability to decreaselight intensity, change spectrum, and/or provide pulsed/intermittentlight to the organism.

Embodiments of the present disclosure can include methods for enhancingphotosynthetic efficiency. An embodiment includes applying light (e.g.,pulsed light of one or more different colors) to a photosyntheticorganism and then using a chlorophyll fluorescence feedback controlsystem to maximize the growth of the photosynthetic organism andmaximize the energy efficiency of the light system (e.g., LEDillumination system). In an embodiment, the chlorophyll fluorescencefeedback control system includes the photosynthetic organism, achlorophyll fluorometer (or similar chlorophyll fluorescence detectionapparatus), and a LED illumination system (e.g., a LED lightingapparatus that can include one or more LEDs at one or more colors (e.g.,white, red, green, orange, green, and blue, light). The chlorophyllfluorometer (or a waveguide or optical fiber to direct the energy to thefluorometer) is disposed adjacent the surface of the photosyntheticorganism to receive fluorescent energy emitted by the photosyntheticorganism, while the LED illumination system directs light energy ontothe photosynthetic organism. The chlorophyll fluorescence feedbackcontrol system measures and monitors fluorescent energy emitted by thephotosynthetic organism (e.g., as a function of time) and uses it togenerate photosynthetic efficiency parameters (e.g., photosyntheticefficiency, photochemical processing, and waste heat dissipation). Oneor more of the photosynthetic efficiency parameters can be used toadjust a pulse rate, pulse on/off duration, light intensity, and/orlight spectrum, of the LED illumination system for drivingphotosynthesis by the delivery of an amount of light to optimize (e.g.,increase) light absorption of the photosynthetic organism, whileproviding enough dark time between light pulses to preventoversaturation of the chlorophyll reaction centers, which can inducephoto-inhibition and photo-oxidation. In order to reduce or preventoversaturation, time between the pulses should be provided so thephotosynthetic organism can re-oxidize the reactions centers. The pulserate, pulse on/off duration, and/or light intensity of one or more ofthe LEDs in the LED illumination system can be adjusted to maximize thelight absorption of the photosynthetic organism and/or reduce the energyused the LED illumination system. Additional details are described inthe Examples.

In an embodiment of the present disclosure, the pulsed light is derivedfrom a LED illumination system. The LED illumination system can includea light source that includes one or more a Light-Emitting Diode (LED) orOrganic Light-Emitting Diode (OLED), where the LED(s) can emit light atdifferent wavelengths. Each of the LEDS in the LED illumination systemcan be operated at a pulse rate of: about 500 Hz, about 1,000 Hz, about2,000 Hz, about 2,500 Hz, about 3,000 Hz, about 3,500 Hz, about 4,500Hz, about 5,000 Hz, about 20,000 Hz, and about 50,000 Hz. In anembodiment, the pulse rate is between about 500 Hz to 10 kHz. Each ofthe LEDS in the LED illumination system can be operated at a wavelengthof: blue LED (440-490 nm), cyan LED (505 nm), white LED (2700 k-10,000k), red LED, green LED, orange/amber LED, and the like, and acombination thereof. Embodiments of the present disclosure can includeone or more Pulse Width-Modulated Light-Emitting Diode (PWM-LED) as asource of one or more of the wavelengths of the pulsed light.

Embodiments of the present disclosure include a biological optimizationsystem where the energy required by the source of pulsed light isdecreased due to the duty-cycle of 50% or less (i.e., the off-timesbetween each LED pulse).

EXAMPLES Example 1 Introduction:

It has long been known that the “Flashing Light Effect” inPhotosynthesis can enhance the light utilization efficiency leading tobetter productivity (Kok, 1953; Phillips and Myers, 1954). The goal isto apply a photon flux density that is just enough to excite themajority of the light harvesting complexes to attain the maximum rate ofgrowth, while simultaneously minimizing trapped surplus light, whichrenders losses in the form of heat and fluorescence. The excess absorbedlight energy can cause damage to the photosynthetic apparatus from thereactive free oxygen radicals generated, known as photoinhibition. Thus,by using intermittent light, the number of excitations arriving at aclosed reaction center decreases when flashes are shortened, permittingmore efficient usage of light and less photodamage repair (Matthijs etal., 1995). The major potential boosts in bioproductivity stems fromimproving flux tolerance rather than from augmenting intrinsicphotosynthesis efficiency. The ultimate rate limiting process forimproving photonic flux tolerance and thus bioproductivity is the timescale for the dark reactions in algal photosynthesis. The matching oftime pattern, spectrum and instantaneous intensity of pulsed LEDphotonic input to the dark reaction kinetics is the key to realizingsuperior flux tolerance (Gordon and Polle, 2007).

The most common physical observable used to assess photosyntheticfunction and its subsequent down regulation in excess-light conditionsis chlorophyll (Chl) fluorescence, because it is sensitive to a widerange of changes in the overall apparatus (Holt et al., 2004). Despitedecades of research on the flashing light effect, there have not beenany studies on the apparent increase in photon utilization efficiency ora minimization of non-photochemical quenching, or heat dissipation usingPAM Fluorometry.

In this Example, PAM Fluorometry has been used to probe PSII underintermittent actinic illumination in order to provide bioenergeticinformation regarding the effective quantum yield, photochemicalquenching and non-photochemical quenching of the photosynthetic organismunder investigation. This research demonstrates that PAM Fluorometry canbe implemented as a diagnostic tool for optimization of photonutilization efficiency and photoinhibition prevention under pulsedactinic illumination for photosynthetically active organisms. Thisresearch has focused on the microalgae, Chlorella sorokiniana andChlorella minutissima as model chlorophyll containing organisms;however, without being bound by any particular theory, it is believedthat this phenomenon will apply to any photosynthetically activeorganism with PSII. This research elucidates that PAM Fluorometrycoupled with a feedback controlled Pulse-Width-Modulated LED actinicillumination system applying various waveform parameters can optimizeartificial illumination for various applications such as high-densityalgal cultivation systems in photobioreactors. The technology developedthrough this research can be used for microalgae cultivation for food,feed, nutraceutical and biofuel production, agriculture, horticultureand coral aquaculture.

Discussion:

The application of intermittent light decreases the number ofexcitations arriving at a closed reaction center, decreasing the need todissipate excess energy as heat, while permitting more efficient use oflight and less photo-damage repair. An objective of this study was toinvestigate the biophysics of the “flashing light effect” under variousintermittent illumination parameters to optimize maximum photonutilization efficiency by using the PAM Fluorometry as a biologicalfeedback mechanism. The biofeedback system will provide a data streamthat can be used to tune the pulse rate and light intensity of the LED'sin order to provide precisely enough light to excite the photosystemwithout over-saturation, thereby minimizing trapped surplus light, whichwill render losses such as fluorescence, excess heat dissipation andpotential oxidative damage to the system.

As the PAR intensity increases, the ability of the LHC's to efficientlyharvest the photon/exciton into effective electron transport diminishes.The fluorescence emitted from the light harvesting complex increaseswith incident PAR, which is indicative of the amount of light saturationachieved within the chloroplast. Continuous light produces morefluorescence, approximately 3 times more at 200 μmol m⁻²s⁻¹ indicatingthat at this light intensity, the majority of the RC's have absorbed onephoton and are now in the semi-open state exhibiting an electrontrapping efficiency of 25%. At higher light intensities, thecontinuously illuminated culture's electron trapping efficiency drops to0% as RC's become completely saturated and closed, whereas the pulsedlight is still able to tolerate the larger flux by still being able totrap 25% of the excitons. Standard PAM protocol uses an induction curvewhich provides a single actinic light intensity over a preset period oftime. This allows measurements of chlorophyll a fluorescence in thesteady state condition over extended exposure. Alternatively, a lightcurve is another method to determine the photosynthetic efficiencies andquenching dynamics of photosystem at various light intensities. Thislight curve can be used to determine the minimal light intensity tosaturate the majority of the LHC's to a semi-open and closed state.

Materials and Methods:

PAM Fluorometer Measurements

The Pulse-Amplitude-Modulation Fluorometer (Mini-PAM, WALZ GmBH,Germany) measurements were performed in real-time using an externalPulse-Width Modulated LED light source as the actinic light sourceduring the PAM measurements of φ_(PSII), qP, NPQ. The culture wascultivated under continuous illumination at 200 μmol m⁻²s⁻¹ and was thendark adapted for one hour before measurements. The measuring lightsource in PAM was a red LED, 650 nm with a standard intensity 0.15 μmolm⁻²s⁻¹ PAR. The halogen light for continuous studies emits a maximum6,000 μmol m⁻² s⁻¹ PAR with continuous actinic illumination and amaximum 18,000 μmol m⁻² s⁻¹ PAR during saturation pulses. The modulationof the measuring light can be set to a modulation frequency 0.6 or 20kHz. For the PWM-LED, the 20 kHz setting was used for the measuringlight frequency in order to attain highest resolution possible of thechlorophyll a fluorescence measurements.

Fluorescence Quenching Analysis

Optimal Quantum Yield: After dark-adaptation.

QY=(Fm−Fo)/(Fm)

Effective Quantum Yield: Under Actinic Illumination.

QY=(Fm′−F)/(Fm′)

Photochemical Quenching: Energy used for the Calvin-Benson Cycle.

qP=(Fm′−F)/(Fm′−Fo′)

Nonphotochemical Quenching: Dissipation of excess light energy in anyway other than through photochemical quenching.

NPQ=(Fm−Fm′)/Fm′

Pulse-Width-Modulated Light Emitting Diode Actinic Illumination System

For these preliminary experiments, a single high powered LED Luxeon K2LED-Royal was driven via a custom built PWM circuit at 475 mW i@ 1000 mAConstant Current using an Agilent E3631A—Triple Output DC Power Supply.The pulse rates used in the experiments were: 500; 1,000; 1,500; 2,000;2,500; 3,000; 3,500; 4,500; 5,000; 20,000; 50,000 Hz. The radiometriclight intensity of the LED was measured by a Li-Cor Quantum Sensor:LI-190. The halogen light used for control was also calibrated usingthis sensor and was set accordingly to output the same amount ofpmoles*m⁻² s⁻¹. The waveform of the PWM circuit was driven by a TTLoutput from a Tektronix AFG3022B-Arbitrary Wave Function Generator, andmonitored by a Tektronix TDS 2022-Digital Oscilloscope.

The frequency and intensity regimes are given in FIG. 6. The light pulsewaveforms offer a particular time sequence for the pulses that persistno longer than tens to hundreds of μseconds, but also as the frequencyincreases, the current draw of the LED also slightly increases providingan elevated instantaneous photon flux for each pulse. Here the averagedphotonic intensity are kept equal among treatments, while postponing theonset of light saturation to higher averaged photonic values. It hasbeen noted that by solely chopping a continuous light signal into pulseswithout increasing the instantaneous photon flux cannot noticeablyimprove bioproductivity, but optimal pulsing can (Gordon and Polle,2007).

Results

Experimental Run: 1

Species: Chlorella sorokiniana

Pulse frequency used

-   -   1 kHz    -   2 kHz    -   5 kHz    -   10 kHz

Control PAR: Halogen-780, 1160 pmoles/m²/s

Culture was dark adapted for 1 hour

Light Induction Curve for 5:00 minutes

C. sorokiniana was used as the experimental organism. The culture wasdark adapted for one hour. The results observed from the induction curveare furnished in FIGS. 7, 8, and 9. FIG. 7 shows the chlorophyll afluorescence measurements for quantum yield or effective photosyntheticefficiency under 455 nm pulsed actinic illumination compared to thewhite halogen light controls providing entire visible range spectrum of780 and 1160 pmoles/m²/s. Chlorella sorokiniana was used at a culturedensity of approximately 0.2 g/L. The treatment of 2 kHz demonstratedsubstantially higher yield values than the controls, as well as a smallbut significant increase over both higher (5, 10 kHz) and lowerfrequency (1 kHz) illumination.

The photochemical quenching of the treatment with 2 kHz frequency inFIG. 7 shows a dramatic increase in the energy undergoingphotoconversion in PSII heading towards carbon fixation compared to thecontrol and other treatments using frequencies 1, 5 and 10 kHz.

FIG. 10 shows results from the first experimental run. The controlhalogen values demonstrated elevated NPQ values, while all of the pulsedactinic treatments had a substantially reduced NPQ or amount of heatdissipated as losses. Most notable is that the 2 kHz treatment had thelargest reduction in wasted energy in accordance with the prediction ofbetween 1-5 kHz as being optimal based on reaction kinetics. The 2 kHzwas pulsed at 50% and thus allows 2 ms⁻¹ dark period for the OEC andplastoquinone dynamics.

Experimental Run: 2

Species: Chlorella minutissima

Pulse Frequency:

-   -   1 kHz    -   1.5 kHz    -   2.5 kHz    -   3 kHz    -   3.5 kHz    -   4.5 kHz

Control PAR: Halogen-780, 1160 pmoles/m²/s

Dark adapted for 1 hour

Light Induction Curve for 15:00 minutes

Chlorella minutissima was the model algae used in the next two sets ofexperiments. The run was conducted for a 15 minute induction curve asopposed to 5 minutes to investigate the steady state of quenchingparameters. This culture was denser (1 g/L) than run 1 with C.sorokiniana. FIG. 10 demonstrates that even more dramatic increases inphotosynthetic efficiency were measured with this treatment. Although asmall difference, the 2 and 3 kHz treatments at 50% duty cycle weremarginally more efficient.

The results from FIG. 11 look almost identical to the yield data,demonstrating again a substantial increase in the amount of chlorophyllexcitations being able to be absorbed by the P680 reaction center andsent towards PSI and carbon fixation in the treatments employing 2 and 3kHz followed by 1, 4.5, 1.5, 2.5 and 3.5 kHz frequencies.

FIG. 12 is a combination of the NPQ data from two experimental runs,which were performed on the same culture within 24 hours of each other.A dramatic decrease in NPQ and losses was observed for the 1, 1.5, 2 and3 kHz treatments. Interestingly, the 2.5, 3.5, and 4.5 kHz treatmentsdid not demonstrate the same minimal NPQ, indicating the possibility forsome sort of non-linear response. The higher frequency treatments of 20and 50 kHz, as predicted, demonstrated high NPQ, drifting up with thecontrols over the 15 minute induction curve. This graph also shows auniversal drift of increasing NPQ for all treatments, indicating thatthe optimal dark periods to minimize qE and excess excitation may varyduring illumination. This drift strongly suggests that a feedback systemwith dynamic self-tuning may provide even more optimal efficiency andNPQ minimization over a fixed frequency pulsed actinic illuminationsource.

Conclusion

The use of Pulse-Amplitude Modulation Fluorometry to assess thebioenergetic state of the photosynthetic organisms under continuous andmodulated illumination demonstrated measurable changes in thefluorescence response of chlorophyll a.

-   -   1. It was found that pulsed LED's can dramatically affect the        Chlorophyll a fluorescence kinetics leading to decreased NPQ,        and enhanced photosynthetic yield and photochemistry.    -   2. Many of the modulated treatments exhibited similar decreases        in excess energy dissipation and increases in photosynthetic        capacity probably due to the similarities in the provided dark        periods. These dark periods allow the dark reactions of        photosynthesis time to process and the reaction centers time to        re-oxidize to a fully open state offering maximal capacity for        exciton capture and electron transfer efficiency.    -   3. The PAM's ability to detect small changes in the way energy        is transferred through photosystem II demonstrates how this        sensitive technique could be used to optimize artificial        illumination for cultivation of plants or algae.    -   4. This study found that the non-photochemical quenching (NPQ)        is optimally minimized by a dark period duration no less than        300 μs, then gradually increase above 1 ms.    -   5. An increase in the effective photosynthetic efficiency        (Yield) of approximately >400% and an additional increase in        photochemical quenching (qP) ranging from ˜200-500% was        exhibited by all samples treated with pulsed light than the        control using continuous illumination.    -   6. These results not only confirm the highly beneficial nature        of the flashing light effect in terms of chlorophyll reaction        kinetics, but also open exciting new applications for optimizing        photosynthesis in real-time with a PAM fluorometric monitoring        system.

Currently, metal halides or high pressure sodium lighting is used as thelight source for photosynthesis in commercial scale agriculture,horticulture and aquaculture cultivation systems. This style of lightinghas many inherent problems, especially for growing photosyntheticallyactive organisms. These high powered lamps are driven by a magnetic orelectronic ballast to generate the controlled currents and required highvoltage. However, the energy losses are substantial due to the heatemitted by the ballasts and bulbs which results in poor efficiency. Thelight produced from metal halide or high pressure sodium is a fullspectrum including green and yellow light which do not directly drivephotosynthesis. However, in a LED system, the optimal actinic spectrumcan be selected where the organism has maximum absorption, which can begenerated and delivered to the light harvesting pigment complexes(LHC's) to increase photosynthetic efficiency. In the case of continuousillumination and high intensity light with full spectrum, a substantialportion of that light will then be dissipated as heat and losses viaNPQ. Thus, the metal halide/HPS/mercury vapor lighting system is aboundwith gross inefficiencies and do not have the capability ofPulse-Width-Modulated (PWM) Light-Emitting-Diode (LED) to optimize thephotonic energies for maximum photoconversion.

REFERENCES

-   Gordon, J. M., and Polle, J. E. W. (2007). Ultrahigh bioproductivity    from algae. Applied Microbiology Biotechnology 76: 969-975.-   Holt, N. E., Fleming, G. R., Niyogi, K. K. (2004). Toward an    Understanding of the Mechanism of Nonphotochemical Quenching in    Green Plants. Biochemistry 42(26): 8281-8286-   Hu, Q., Zarmi, Y., Richmond, A. (1998). Combined effects of light    intensity, light path and culture density on output rate of    Spirulina platensis (cyanobacteria). European Journal of Phycology    33:165-171.-   Katsuda, T., Shimahara, K., Shiraishi, H., Yamagami, K., Ranjbar,    R., and Katoh, S. (2006). “Effect of Flashing Light from Blue Light    Emitting Diodes on Cell Growth and Astaxanthin Production of    Haematococcus pluvialis.” Journal of Bioscience and Bioengineering    102(5): 442-446.-   Kim, Z.-H., Kim, S-H., Lee, H-S., Lee, C-G. (2006). “Enhanced    production of astaxanthin by flashing light using Haematococcus    pluvialis.” Enzyme and Microbial Technology 39: 414-419.-   Kok, B. (1953). In: Algal Culture: From Laboratory to Pilot Plant.    Ed: J. S. Burlew. Carnegie Inst. Washington Publ. 600. Chapter 6:    63-75.-   Matthijs, H. C. P., Balke, H., van Hes, U. M., Kroon, B. M. A.,    Mur, L. R., Binot, R. A. (1995). “Application of light-emitting    diodes in bioreactors: Flashing light effects and energy economy in    algal culture (Chlorella pyrenoidosa).” Biotechnology and    Bioengineering 50 (1): 98-107.-   Mauzerall, D. (1972). Light-Induced fluorescence changes in    Chlorella, and the primary photoreactions for the production of    oxygen. Proceedings of the National Academy of Science, USA 69:    179-189.-   Müller, P., Li, X.-P., Niyogi, K. K. (2001). Non-Photochemical    Quenching, A Response to Excess Light Energy. Plant Physiology 125:    1558-1566.-   Park, K.-H., and Lee, C-G. (2000). “Optimization of Algal    Photobioreactors using Flashing Lights.” Biotechnol. Bioprocess Eng.    5: 186-190.-   Phillips, J. N. and Meyers, J. (1954). Growth Rate of Chlorella in    Flashing Light. Plant Physiology 29(2): 152-161.-   Richmond, A., Zhang, C. W., Zarmi, Y. (2003). Efficient use of    strong light for high photosynthetic productivity:    interrelationships between the optical path, the optimal population    density and cell-growth inhibition. Biomolecular Engineering    20:229-236.-   Terry, K. L. (1986). Photosynthesis in modulated light: quantitative    dependence of photosynthetic enhancement on flashing light rate.    Biotechnology and Bioengineering 28: 988-995.-   Vrenenberg, W. J. (2004). System Analysis and Photochemical Control    of Chlorophyll Fluorescence in Terms of Trapping Models of    Photosystem II: A Challenging View. George C. Papageorgiou and    Govindjee (eds): Chlorophyll a Fluorescence: A Signature of    Photosynthesis, Springer, pp. 133-172.-   Wijn, R. d. and Gorkom, H. J. v. (2001). Kinetics of Electron    Transfer from Q_(A) to Q_(B) in Photosystem II. Biochemistry 40:    11912-11922.

Example 2 Abstract

The experimental results of the microalgae, Chlorella sorokiniana,chlorophyll a fluorescence analysis on three different growth stagesidentified at its Optical Density (OD) at wavelength 735 nm OD: 0.5, 1.0and 1.5. The cultures were irradiated with 6 monochromatic LightEmitting Diodes (LED) and three full light spectrum white LEDs (neutralwhite, cool white and warm white). The chlorophyll fluorescenceparameters of quantum yield of Photosystem II (φ_(PSII)) andNon-photochemical quenching (NPQ) were measured for each sample.Absorbance spectra curves were scanned using C. sorokiniana in vivo atthe three establish ODs. An inverted correlation between φ_(PSII) andlight intensity was found based on light absorption of C. sorokinianapigment content, which is rich in the pigment lutein. Due to changes inabsorption at various wavelengths, C. sorokiniana exhibited a lowerφ_(PSII) in the green spectrum over the red spectrum of light as theculture increased in OD. NPQ had a noticeable decrease as the culturemoved from OD 0.5 to 1.0 due individual cell's exposure to lowerphotosynthetic photon flux density (PPFD).

Introduction

Microalgae cultivation for biofuels has been praised as a promisingfeedstock for conversion into liquid transportation fuels. However, thepetroleum industry exhibits competitive pricing and market fluctuations,which make it hard for potential microalgae producers to compete forlow-value transportation fuels. Traditionally, the cultivation ofmicroalgae has been reserved for use as a high protein “green food”supplement as well as a feedstock for high value bioactive compounds andnutrients. These high value bioproducts obtained from various species ofmicroalgae and cyanobacteria can offer much higher market value at muchlower quantities than biofuels (1). The development of methods to inducehigher biomass production is of paramount importance for microalgaecultivation for biotechnology and bioactive compounds. Despite highercapital costs, photobioreactors (PBR) are the best way to achieve higherbiomass production and maintaining monoculture by being able tospecifically control various environmental parameters. The higher costsassociated with PBR's and artificial illumination can only be warrantedby high value products and high growth productivity. There are a severaldesigns for PBR's which attempt to optimize mass transfer, lightexposure, and environmental control of the culture (2). Even in theseoptimized systems, the irradiance onto the cells is still one of themajor limiting factors. As the culture achieves high density population,the light penetration substantially decreases due to physical shadingand physiological factors described here.

Incident radiation with wavelengths of 400-700 nm is generallyconsidered the photosynthetically active component of the total spectralirradiance and is termed Photosynthetically Active Radiation (PAR) (3).All kinds of lamps can provide PAR, however, the energy conversionefficiency from electric energy to visible light energy variesdramatically depending on the light source. Lighting efficiency ismeasured in light output per watt and represents the amount of lightproduced for each watt of electricity consumed. Incandescent and halogenlights have the lowest lighting efficiency (10-17 and 12-22 lumens perwatt, respectively) whereas linear fluorescent and high pressure sodiumhave higher efficiency (30-110 and 50-140 lumens per watt,respectively). Although useful for comparison, the light output here isgiven in the photometric unit, lumens, which give higher values to lightspectra with more green. However, plant photosynthesis is drivenprimarily by blue and red portions of the spectrum, which are notequally represented by a lumen rating.

By providing plants with full spectrum PAR, a portion of the electricenergy converted to light is wasted for photochemical conversion in theorganism as a consequence of light reflection and/or heat dissipationthrough biochemical pathways, such as the xanthophyll cycle. Theillumination of a culture by specific wavelengths which match thespectral absorbance of the microalgae pigments can avoid these energylosses by achieving a better growth rate/energy consumption ratio. LightEmitting Diodes (LED) can emit light at specific wavelength ranges, thusit is possible to drive photosynthesis by providing only the mosteffective wavelengths to various microalgae species accordingly to theirpigment profile. LED systems can also provide pulsed light where burstsof high irradiance are followed by a short dark period to allow time forthe photochemical processes to restore the chlorophyll reaction center.Experiments with pulsed LED's have been able to sustain near maximumgrowth rates with major gain in energy efficiency while exhibiting lowerheat losses compared to fluorescent lighting (4). The advancements inLED technology are now making commercially available LED's with lightefficiencies in the range of 80 and recently as high as 200 lumens perwatt (5). In addition, the energy converted by LEDs will be emitted asspecific wavelengths that can be tuned for photosynthetic absorptionthereby decreasing energy wasted from reflection and internal lossesunder full spectrum light.

The Photosynthetic process begins at light absorption by thechloroplast. Absorbed photons are trapped by chlorophyll and otherpigment's antennae complex embedded in the thylakoid membrane of thechloroplasts. Pigments are arranged in three dimensional pigment-proteincomplexes which determine their function and efficiency of energytransfer. The spectrum of light absorbed is determined by the pigmentcomposition of each specific photosynthetic organism. Each pigment has aspecific light absorption capacity, and varies by both the spectrumabsorbance as well as its absorption coefficient. The main pigmentsfound in terrestrial plants, microalgae and cyanobacteria arechlorophyll a and b with maximum light absorption peaks in the blue andred spectrum; and carotenoids with light absorption peaks in the blueand yellow spectrum.

The light absorbed by antennae complex has three fundamental fates: itis either used to drive photosynthesis (photochemical quenching—qP);dissipated as heat (Non-photochemical quenching—NPQ); or re-emitted asfluorescence. The energy directed to the reaction centers P680 (RCII) ofPhotosystem II (PSII) and P700 of Photosystem I (PSI) is used to drivethe light reactions. At RCII water molecules are split releasingelectrons to the electron transport chain. Those electrons are conductedthrough a series of acceptors to PSI where it is excited again and usedto produce the reductant molecule NADPH in a process named ‘Z’-schemefirst described by Hill and Bendall (6). During this process, H⁺ protonsreleased from the splitting of water plus H⁺ protons pumped across thelumen membrane accumulate inside the lumen creating a proton gradientwhich drives ATP synthesis. Protons escaping from the thylakoid lumenthrough a central core of the enzyme ATP synthase cause conformationalchanges in the enzyme which catalyzes the phosphorylation of ADPproducing ATP on the stromal side (7). Both ATP and NADPH molecules arefurther used to reduce CO₂ molecules to hexose in the Calvin-BensonCycle (dark reactions). The dark reactions are slower than lightreactions, so under high light conditions the electron flow is saturatedand the electrons transported on light reactions can be accepted byother molecules rather than NADP⁺ (e.g., O₂) producing free radicals.These highly reactive molecules can cause photodamage andphoto-oxidation to the photosynthetic apparatus mechanism causingreversible photoinactivation or in severe conditions permanentinactivation of PSII reaction centers, known as photoinhibition (8).

Microalgae, as all the other higher plants, have protective mechanismsthat dissipate the excess of absorbed light energy as heat (NPQ). Themost effective and fastest NPQ mechanism is termed high energy statequenching (qE). This process is induced by the pH decreasing in thelumen and causes the protonation of PSII proteins activating thexanthophyll synthesis within the carotenoids pigments via xanthophyllcycle. At low lumen pH, the enzyme violaxanthin de-epoxidase removes twoepoxides groups from the violaxanthin transforming this pigment into azeaxanthin. The interaction of zeaxanthin with chlorophyll dissipatesexcitation energy as heat rather than transferring it to chlorophyllmolecules.

Fluorescence is the re-emission of energy in the form of a photon as anelectron returns to ground state from a singlet excited state. As someenergy is also given off as heat, the photon is red-shifted with anemission peak of ˜685 nm (3). At room temperature nearly allfluorescence (90-95%) comes from PSII at 685 nm. Fluorescence emitted byPSI is very low at normal temperatures and only can be detected at thetemperature of liquid nitrogen. Indeed, the primary acceptor ofelectrons from P700 is rapidly re-oxidized, reducing fluorescenceemissions at PSI (9).

The maximum rate of photosynthesis is independent of wavelength (10),however, it is important to find the maximum light intensity thatprovides maximum biomass production at each specific wavelength withoutproducing damage-reactive species (e.g., ¹O₂*) as byproducts ofphotosynthesis (11). Finding the best LED wavelengths at optimalintensities will optimize photochemical conversion instead of NPQ.

Fluorescence can be used to estimate the quantum efficiency of chargeseparation at RCII (12). The use of fluorescence measurements as a probeof photosynthetic productivity have been vastly investigated due to thesimplicity of the measurements. Many studies correlating photosyntheticcarbon fixation and electron transport rate based on changes influorescence yield have been reported (13) (14) (15). However, theunderlying theory and the interpretation of data remains complex.

In the present disclosure, fluorescence re-emissions were monitoredusing a PAM fluorometer from the microalgae C. sorokiniana at differentgrowth stages to analyze how the proportion of light absorbed bypigments associated with PSII used to drive photosynthesis (quantumyield of Photosystem II-φ_(PSII)) and dissipated as heat (NPQ) areaffected by six different monochromatic LED's and three differenttemperature rated white LED's compared at three different lightintensities and three culture densities.

Materials and Methods

Algal Culture

Chlorella sorokiniana (UTEX 2805) was obtained from UTEX CultureCollections and maintained in BG11 medium (NaNO₃, 17.6 mM; K₂HPO₄, 0.22mM; MgSO₄.7H₂O, 0.03 mM; CaCl₂.2H₂O, 0.2 mM; citric acid.H₂O, 0.03 mM;ammonium ferric citrate, 0.02 mM; Na₂EDTA.2H₂O, 0.002 mM; Na₂CO₃, 0.18mM; H₃BO₃, 46 μM; MnCl₂.4H₂O, 9 μM; ZnSO₄.7H₂O, 0.77 μM; Na₂MoO₄.2H₂O,1.6 μM; CuSO₄.5H₂O, 0.3 μM; Co(NO₃)₂.6H₂O, 0.17 μM). The pH value ofculture medium was adjusted to 7.0±0.2 before inoculation and the algaewere maintained in a temperature controlled illuminated growth chamberat 25±1° C. and 100±10 μmoles/m²/s light intensity provided by coolwhite fluorescent (6500K) T-8 bulbs.

Samples Growth

The microalgae cultures were grown in BG11 nutrient media. For thepreparation of the mother culture, two flasks were filled with 270 mL ofBG11 media and inoculated with 30 mL's of C. sorokiniana (10% by volume)in exponential phase. The flasks were placed into a temperaturecontrolled illuminated growth chamber. Samples from both flasks weretaken daily for optical density measurements at 750 nm. The samples weregrown to three specific optical densities, i.e., OD₇₅₀: 0.5, 1.0, 1.5.Once the samples reached the desired density, the culture was removedfrom the growth chamber and was then used in the LED-PAM experiments.

Optical System

Two LED panels were built using aluminum heat sinks. The first LED panelcontains five Luxeon® III Star LED's: red (λ_(627nm)), red-orange(λ_(617nm)), amber (λ_(590nm)), green (λ_(530nm)), cyan (λ_(505nm)) andone Luxeon® K2 Emitter LED: blue (λ_(470nm)). The second panel containsthree Luxeon® K2 Emitter LED with temperature ratings of neutral white(5000K), cool white (6500K) and warm white (2700K). Each LED was used toilluminate the microalgae samples through a ¼ inch diameter fiber-opticlight guide positioned 1 mm away from the surface of the 10 mmpath-length cuvette. A light condenser was attached from the flat end ofthe fiber optic cable to the LED to collect and direct the LED outputthrough the light guide. Each LED was driven by a single Luxdrive™ 1000mA Buckpuck 3021-D-E-1000. The Buckpuck driver was powered by an Agilentpower supply (Agilent Technologies, Santa Clara, Calif., USA) whichenables voltage and current regulation to adjust light intensity output.Radiometric light intensity measurements were performed at tip of thefiber optic light guide and reported in μmol m⁻² s⁻¹ using a quantumsensor (LI-COR, Quantum Sensor LI-190, and Datalogger, LI-1400 Lincoln,Nebr., USA).

Analytical Methods

Absorbance spectrum from 400-800 nm was measured, and the cultureoptical density was recorded at 750 nm using a Varian Cary 50 UV/Visiblespectrophotometer (Varian, Inc, Santa Clara, Calif., USA). Thechlorophyll a fluorescence parameters, quantum yield of Photosystem II(φ_(PSII)) and non-photochemical quenching (NPQ), were determined by aPulse Amplitude Modulation (PAM) fluorometer Walz Mini-PAM (Heinz WalzGmbH, Effeltrich, Germany). The microalgae samples were analyzed insidea 5.0 mL cuvette with 10 mm path-length. Two fiber optic light guideswere positioned on the surface of the cuvette at right angles. One lightguide was connected to the LED panel for illumination and the secondlight guide was connected to the Mini-PAM Fluorometer for fluorescencemeasurements. The cuvette and light guides were contained inside ahinged-top dark enclosure (12.5 cm×15.5 cm×16.5 cm) custom made out ofdelrin plastic.

Experimental Procedure

Samples at three different optical densities were each illuminated byone of the nine LED's at three different light intensities (100, 250 and500 μmol m⁻² s⁻¹), for 5 minutes while Chlorophyll a fluorescence wascontinuously monitored. All treatments were carried out in duplicates.

In order to record maximum fluorescence yield, samples were dark adaptedfor 30 minutes prior to fluorescence analysis to allow total NPQrelaxation and fully oxidize Q_(A). 3 mL aliquots were placed at thecuvette for each run, following the process described by Maxwell andJohnson (16) both φ_(PSII) (Eq. 1) and NPQ (Eq. 2) were recorded. Thesaturating light pulses from the PAM's internal halogen bulb had aduration of 1.5 seconds at 10,000 μmol m⁻² s⁻¹, and were used to producea transient closure of the PSII photochemical reaction center every 20seconds for a 5 minutes run:

φ_(PSII)=(F′ _(m) −F _(t))/F′ _(m)  Eq. 1

NPQ=(F ^(o) _(m) −F′ _(m))/F′ _(m)  Eq. 2

where F′_(m) is the fluorescence maximum in the light; F_(t) is thesteady state value of fluorescence immediately prior to the flash andF^(o) _(m) is the maximum fluorescence value in dark-adapted state,obtained by simultaneously exhibition to the actinic (LED) and thesaturating light.

The treatments were defined by a LED color, light intensity and culturedensity. Each treatment had a fresh 3 ml of algal culture injected intothe sample cuvette and the φ_(PSII) and NPQ measurements were taken for5 minutes to produce the induction curve. An average of the last threemeasurements of φ_(PSII) and NPQ were taken as the final equilibriumvalues for each treatment. The final results reported in the tables werecalculated from the average of duplicate runs per treatment. Statisticalanalysis was performed using SAS 9.2. The φ_(PSII) data was analyzed asa Factorial Design within each ODs and NPQ was analyzed as a FactorialDesign within each LED color.

Results

Quantum Yield

Among the three white LEDs (neutral white, cool white and warm white),the neutral white LED always exhibited the best φ_(PSII) (p<0.05). Tofacilitate the visualization of the data for full spectrum white light,the results from the three white LED's are reported together. Ingeneral, as the light intensity increases, a decrease in φ_(PSII) valueswas observed for all monochromatic wavelengths and the full spectrumwhite LED (Table.1). Orderly interaction was observed between thefactors of light intensity and color at an OD of 1.0 and 1.5. Despitedifference in the magnitude between levels of light intensity changefrom monochromatic wavelengths and white LED, the order of means forlevels of light intensity is always the same. Thus, the main effects ofwavelength and light intensity are meaningful.

At the low density culture (OD 0.5) λ_(470nm) had lower φ_(PSII) andλ_(505nm) had higher φ_(PSII) compared to the control white LED whereasall the other wavelengths showed no significant difference. At themedium density (OD 1.0) λ_(470nm) and λ_(617nm) had lower φ_(PSII)compared to control white LED which had no significant difference toλ_(627nm). All the other wavelengths had a higher φ_(PSII) compared tocontrol white LED. At higher culture density (OD 1.5) λ_(470nm) andcontrol white LED showed the lower φ_(PSII) with no significantdifference, whereas all the other wavelengths had higher φ_(PSII).

Within the monochromatic wavelengths, λ_(530nm) and λ_(505nm) exhibitedhigher φ_(PSII) on samples with OD 0.5, whereas on samples with OD 1.0showed a gradual drop in φ_(PSII) switching position with λ_(590nm),λ_(617nm) and λ_(627nm) on samples with OD 1.5. At the red spectrum(λ_(617nm); λ_(627nm)), φ_(PSII) does not drop as the samples ODincrease from 1.0 to 1.5 in any of the 3 light intensities (shaded areaon Table.1). For λ_(590nm) the variation in φ_(PSII) as OD increasesfrom 1.0 to 1.5 is very small.

TABLE 1 Results for Quantum yield of Photosystem II (Φ_(PSII)) of C.sorokiniana at three Optical Densities (OD) under interactive Wavelength(λ) and Light intensity. Φ_(PSII) Light intensity λ (nm) OD (μmol m⁻²s⁻¹) 470 505 530 590 617 627 White 0.5 100 0.4387 0.6388 0.5575 0.54850.4910 0.5357 0.5492 250 0.3700 0.6378 0.5428 0.4513 0.4592 0.47630.4815 500 0.3255 0.5352 0.4995 0.4398 0.3612 0.4452 0.4433 1 100 0.32080.3770 0.3983 0.3540 0.2981 0.3080 0.3700 250 0.2340 0.3012 0.35900.2853 0.2477 0.2476 0.2940 500 0.1365 0.2286 0.3136 0.2198 0.16430.1966 0.2041 1.5 100 0.1995 0.2252 0.2495 0.3055 0.3028 0.3202 0.2097250 0.1402 0.1718 0.2228 0.2568 0.2445 0.2717 0.1587 500 0.0793 0.13450.1893 0.2102 0.1585 0.2183 0.1150 Significant Effects (P < 0.001) ODLight Intensity color Light intensity × color 0.5 25.37 29.38 n. s. 1.0838.48 174.44 11.95* 1.5 428.44 234.43 8.98* * refers to orderlyinteraction. Shaded part indicates the constant Φ_(PSII) values.

Non-Photochemical Quenching

Non-Photochemical Quenching (NPQ) refers to the difference between theinitial, dark-adapted maximum level of fluorescence and that recordedafter a period of illumination (17). Although NPQ does not show anylogical behavior when analyzed within each OD either as a lightintensity or wavelength response (model p-value >0.05), a relation wasfound when it was analyzed within wavelengths as culture OD response(Table 2). As the culture increased its OD from 0.5 to 1.0 NPQ had asignificant decrease at all monochromatic wavelengths and the controlwhite LED. At λ_(470nm) an interaction between the factors: lightintensity and OD, was observed. Using T comparison lines for leastsquare means of the interaction OD*light intensity we identified thesame significant drop in NPQ from OD 0.5 to 1.0 (shaded area Table 2).

TABLE 2 General Linear Model for Non-photochemical quenching (NPQ) of C.sorokiniana at six monochromatic wavelengths (λ) and full spectrum whiteLEDs under interactive Optical Densities (OD) and Light Intensity. NPQLight Intensity λ (nm) OD (μmol m⁻² s⁻¹) 470 505 530 590 617 627 White0.5 100 0.2393 0.2490 0.1932 0.3463 0.1853 0.2383 0.1685 250 0.29350.1992 0.2103 0.2388 0.1663 0.2220 0.2033 500 0.3858 0.3102 0.16720.2380 0.2412 0.1842 0.1890 1 100 0.0703 0.0360 0.0633 0.0477 0.13420.1435 0.0580 250 0.1432 0.0605 0.0715 0.0772 0.1545 0.1480 0.0983 5000.1078 0.1363 0.0950 0.1315 0.1732 0.1113 0.1432 1.5 100 0.0932 0.06920.0638 0.0990 0.0447 0.0893 0.1103 250 0.0923 0.0705 0.0757 0.08470.0430 0.0810 0.1133 500 0.0863 0.0950 0.0685 0.0715 0.1432 0.07230.1260 Significant Effects OD 108.34*** 31.8*** 50.37*** 13.5** 26.87***26.36*** 15.53** Light Intensity 6.95* 4.64* n.s. n.s. 9.39** n.s. n.s.Light intensity*OD 5.08* n.s. n.s. n.s. n.s. n.s. n.s. * refers tosignificance at P < 0.05; ** to significance at P < 0.01; *** tosignificance at P < 0.001. Shaded part indicates the significantdifferent NPQ values.

Light Absorption Spectra Curves

Measurements of light spectrum absorbance were taken on C. sorokinianasamples at OD 0.5, 1.0 and 1.5. All wavelengths demonstrated higherabsorption at denser cultures (FIG. 14A). Apart from variations inamplitude, wavelengths that overlap the absorbance spectrum ofcarotenoids and chlorophyll (λ_(470nm) and λ_(505nm)) showed constanthigher absorbance than the other wavelengths. A valley between 550 nmand 650 nm and a peak at 680 nm can be observed (FIG. 14A). The oldersamples at OD 1.5 (30 days) and OD 1.0 (15 days) diluted to OD 0.5 werescanned and compared to the youngest sample at OD 0.5 (7 days). Theolder samples showed less absorption within PAR spectrum than the youngsample (FIG. 14B).

Discussion

To increase the energy efficiency of cultivating microalgae and othergreen-house cultures using artificial supplemental lighting, manystudies have been conducted to find the best wavelengths, intensitiesand frequencies of light that can induce the maximum photochemistry withminimum NPQ. Low light intensities illumination may not induce thenecessary photochemistry to maximize biomass production. Whereas highlight intensities may sufficiently drive photochemistry but can resultin both wasted energy as heat and photodamage of chloroplast (8).

Microalgal biomass yields higher than 100 g dry weight m⁻² d⁻¹ areprojected under optimal conditions, which can only be provided usingLED's due their temporal, spectral and intensity characteristics (18),related to the balance of the rates at which the photosynthetic electronflow from PSII reduces Q_(A) and the efflux toward PSI which oxidizesQ_(A) (4).

As the microalgae cultures in ponds increase in density, lightpenetration becomes a major issue to sustain high growth productivities.Without being bound by any particular theory, we suspect thatwavelengths with lower absorption by the primary pigments (chlorophyll,carotenoids, etc), can penetrate deeper into dense cultures and offerbetter light distribution in the pond. Colors such as green, whichexhibit a high reflection coefficient, can homogenously distribute lightenergy at deeper levels where the other more strongly absorbedwavelengths, such as blue and red, become filtered out.

Light Absorption

Chlorella sorokiniana is rich in carotenoid pigments (0.69% of drymatter) that can absorb light in the blue and green spectrum of PAR (400nm to 530 nm). Lutein is the primary carotenoid pigment found in C.sorokiniana representing 60% of the total carotenoids (19) (Table 3).

TABLE 3 C. sorokiniana carotenoid pigments content and its maximumabsorbance wavelength corrected for acetone (λ_(ad)). ^(a)ContentCompound (μg g⁻¹ dry weight) ^(b)λ_(ad)[nm] Carotene 600 452Cryptoxanthin 36 452 Lutein 4300 448 Zeaxanthin 140 452 TotalCarotenoids 6900 ^(a)taken from Matsukawa et al (2000); ^(b)taken fromBiehler et al (2010)

β-carotene (not listed in Table 3), also called the red-protein, isanother carotenoid pigment present on C. sorokiniana. It is present at600 μg g⁻¹ dry weight (19) and was reported to have peak absorptionspectrum at 538 nm on spinach lamellae (20) and parsley leaves (21). Infreeze-dried chloroplasts from wheat leaves 8-carotene and Lutein showedpeak absorptions at 510 nm and 495 nm (22). Specific absorptioncoefficients of 2144 and 403.3 (100 mL g-1 cm⁻¹) at λ_(472 nm) andλ_(508nm) respectively were found for the yellow isochromic fraction(β-carotene, β-cryptoxanthin and zeaxanthin) of paprika and red pepperoleoresins carotenoids (in acetone) (23).

Total carotenoids in the batch cultivation of microalgae follow the samepattern as the biomass productivity, showing growth at an exponentialrate and reaching the steady state along with cell biomass. However, thecarotenoid content measured in the individual cells decreases with time(24). The same pattern is observed for chlorophyll a and b pigments inbatch cultures growth. Different age samples diluted to the same ODshowed differential absorption at PAR spectrum of light indicating lesspigments in older individual cells than younger cells (FIG. 14B).

Total extracted pigment curves containing chlorophyll a and b andcarotenoids present high absorbance at the blue and red spectrum. Thehigher absorbance in the blue spectrum is attributed to an overlap inprimary chlorophylls and carotenoid absorption peaks. Although the twored spectrum LEDs λ_(617nm) and λ_(627nm) are absorbed exclusively bychlorophyll present in C. sorokiniana these two wavelengths fall out ofchlorophyll a and b main absorbance peak at λ_(680nm) and λ_(660nm)(25). Despite the higher number of cells in samples at OD1.5 compared tosamples at OD1.0, no change in φ_(PSII) was observed under any of thethree light intensities at these two wavelengths (λ_(617nm) andλ_(627nm)).

Yield

Changes in fluorescence parameters analyzed here (φ_(PSII) and NPQ)should not be attributed to photodamage of the photosynthetic apparatus.The light intensities used in this work were not high enough to saturatethe electron flow. Wang, et al. (26) tested the effects of differentLEDs on Spirulina platensis found that biomass production increases withincreasing intensities from 300 to 3000 μmol m⁻² s⁻¹ using monochromaticλ_(627nm), λ_(470nm) and λ_(530nm) and full spectrum white LEDs.

In this research, we found an inverse correlation between lightintensity and φ_(PSII). The overall decrease in φ_(PSII) is associatedto more efficient use of energy at low irradiations. Second Sugget etal. (12), φ_(PSII) here defined can be de-composed into:

φ_(PSII)′=φ_(P)′×φ_(NPQ) ^(′)

These measurements are taken under actinic light illumination, indicatedby the apostrophe. φ_(P)′ estimates the efficiency of charge separationin RCII and φ_(NPQ)′ is defined as the efficiency of excitation energytransfer from antenna pigments to the RCII. Any NPQ of PSII will reduceφ_(PSII)′. Providing algae with higher light intensities more light isabsorbed and more RCII will be reduced causing a decrease in φ_(P)′value with a consequently dropping on φ_(PSII) value. As defined, yieldis the part of light absorbed that promotes photosynthesis. Increasingthe total amount of light absorbed cause a natural decrease inefficiency charge separation on RCII promoting more NPQ leading to adecrease on φ_(PSII)′. Analyzing the individual parameters F′_(m) andF_(t) (used to calculate φ_(PSII) on Eq. 1) Genty et al. (15)demonstrated larger drops in F′_(m) compared to the relatively stablevalues of F_(t) during increasing light intensities from 0 to 500 μmolm⁻² s⁻¹, which leads to an overall φ_(PSII) decreases. Fluorescenceanalysis conducted on phytoplankton samples before and after incubationat fixed depth showed a maximum reduction in operational quantum yieldof 34% on samples exposed to full solar irradiance (water surface)whereas 2 meter depth samples showed an increase of 23% when compared totheir corresponding values before incubation (27). Exposing the samplesto higher PPFD caused a decrease on φ_(PSII) as a consequence of morelight absorption by the whole culture, reducing more RCII decreasing theefficiency of photochemical quenching under saturating light pulses.

Wavelengths

The strongest absorbed LED, λ_(470nm), showed the lowest yield onsamples at all OD's tested. In high density cultures, less than 1% ofthe blue light is transmitted through the chloroplast, thus allλ_(470nm) light emitted on microalgae culture is readily absorbed by thecell layers at the surface of illumination on the cuvette, which isdifferent from the other wavelengths tested that can have morereflection than absorption. Pickett and Myers (10) comparingmonochromatic light saturation curves for photosynthesis in Chlorellafound different monochromatic light saturation points for wavelengths onblue, red and green spectrum. Despite the fact that the maximum rate ofphotosynthesis is independent of wavelength within PAR spectrum to reachthe same photosynthetic rate at λ_(630nm) (weakly absorbed band) it wasnecessary to provide 5 times the light intensity used at λ_(450nm)(strongly absorbed band) due differential amount of light absorbed.Providing different wavelengths at the same light intensity will cause adifferential absorption of energy. The low amount of energy absorbed atλ_(530nm) at OD 0.5 is fully drained by the available oxidized reactioncenters, whereas at strongly absorbed wavelengths, the increased amountof energy absorbed naturally reduces more reaction centers suffering aconsequential drop on φ_(PSII).

In the present disclosure, φ_(PSII) of the λ_(505nm), λ_(530nm) andwhite LED showed a significant decrease compared to the otherwavelengths as the culture increases its density, changing position withλ_(590nm) and λ_(627nm) in the stationary phase culture (OD1.5). Itsuggests the better absorption by carotenoids in the blue and greenspectrum of light, whereas the amount of chlorophyll increasing was notsufficient to show a significant increase of the weakly absorbedλ_(590nm) and λ_(627nm). Green and cyan light which can penetrate deeperinto the sample is better distributed inside the cuvette allowing betterand more homogenous absorbance by the carotenoids pigments present on C.sorokiniana. Emerson and Lewis (28) illuminated a thick layer ofChlorella cells with wavelengths in the blue (λ_(450nm)), green(λ_(550nm)) and red (λ_(600nm)) and found scattered indices by greenlight 2 times higher than blue and 1.65 times higher than red. Thus,these weakly absorbed wavelengths at higher OD culture are lessabsorbed, but more homogeneously distributed than red light in the wholeculture, without saturating the reaction centers. White LED showed thelowest φ_(PSII) due its increased absorption at blue and green spectrum.

NPQ

Past studies measuring quantum yield as oxygen evolved by photosynthesisbased on light incident do not take into account NPQ as one of thepossible fates of absorbed light. NPQ is a complex mechanism and adifficult parameter to be understood. The drop in NPQ from samples at OD0.5 to OD 1.0 is a response to more efficient light absorption insidethe cuvette caused by self-shading as a consequence of more cellspresent at the sample, independent of wavelength or light intensity. Thelower concentration of cells present at OD 0.5 were exposed to an excessof photosynthetic photon flux density (PPDF) saturating thephotosynthetic electron transport. Samples with higher OD contain moreindividual cells which are exposed to reasonable levels of lightachieving more efficient energy absorption overall. Intact colonies offilamentous cyanobacteria Oscillatoria showed much less susceptibilityto photoinhibition than individual trichomes at the same high lightirradiance due to the increased specific absorption coefficient causedby a reduction in self-shading (29). Increased C. sorokiniana OD causesimilar self-shading effect observed on filamentous colonies, reducingNPQ.

Conclusion

The experimental results from the quantum yield data and lightabsorption spectra of C. sorokiniana samples at three specific ODindicate an inverted correlation between light intensity and chlorophylla fluorescence yield (φ_(PSII)) measured at specific monochromaticwavelengths as well as the full spectrum white LED. Light intensityincreasing from 100 to 500 μmol m⁻² s⁻¹ can significantly changeφ_(PSII) emissions without significantly impacting NPQ.

Wavelengths absorbed by carotenoids can provide extra φ_(PSII) drop indenser algal cultures when wavelengths weakly absorbed exclusively bychlorophylls are no longer capable of penetrating as deep into theculture to be absorbed by interior cells. The lower yields observed atthe blue light spectrum represent the high energy absorption atλ_(470nm) by C. sorokiniana pigment content. Wavelengths on red spectrum(λ_(590nm), λ_(617nm) and λ_(627nm)) did not have a good absorption bythe main pigments chlorophyll a and b. As the culture became densermoving from OD 1.0 to 1.5 no changes on φ_(PSII) were observed.

The highest light intensity source (500 μmol m⁻² s⁻¹) was the bestoption for LEDs which can provide specific wavelengths at the bestspectrum with both economic and photosynthetic energy efficiency.Without being bound by any particular theory, it is suspected that athigh density culture (OD>2) increasing light intensity can significantlyincrease the portion of the light absorbed as well as the part used todrive photosynthesis from weakly absorbed wavelengths with nosignificant NPQ increases. Whereas, the same increase in light intensityof strongly absorbed wavelengths, such as blue and red will notsignificantly change φ_(PSII), but will increase NPQ. Further studiesshould be conducted using higher light intensities applied to cultureswith higher ODs.

REFERENCES

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It should be noted that ratios, concentrations, amounts, and othernumerical data may be expressed herein in a range format. It is to beunderstood that such a range format is used for convenience and brevity,and thus, should be interpreted in a flexible manner to include not onlythe numerical values explicitly recited as the limits of the range, butalso to include all the individual numerical values or sub-rangesencompassed within that range as if each numerical value and sub-rangeis explicitly recited. To illustrate, a concentration range of “about0.1% to about 5%” should be interpreted to include not only theexplicitly recited concentration of about 0.1 wt % to about 5 wt %, butalso include individual concentrations (e.g., 1%, 2%, 3%, and 4%) andthe sub-ranges (e.g., 0.5%, 1.1%, 2.2%, 3.3%, and 4.4%) within theindicated range. The term “about” can include ±1%, ±2%, ±3%, ±4%, ±5%,±6%, ±7%, ±8%, ±9%, or ±10%, or more of the numerical value(s) beingmodified. In an embodiment, the term “about” can include traditionalrounding according to significant figures of the numerical value. Inaddition, the phrase “about ‘x’ to ‘y’” includes “about ‘x’ to about‘y’”.

It should be emphasized that the above-described embodiments of thepresent disclosure are merely possible examples of implementations, andare merely set forth for a clear understanding of the principles of thedisclosure. Many variations and modifications may be made to theabove-described embodiments. All such modifications and variations areintended to be included herein within the scope of this disclosure andprotected by the following claims.

1. A method for enhancing photosynthetic efficiency comprising: applyingpulsed light to a photosynthetic organism; using a chlorophyllfluorescence feedback control system to determine one or morephotosynthetic efficiency parameters, wherein the photosyntheticefficiency parameters are used to adjust one or more of the following: apulse rate, pulse on/off duration, light intensity, light spectrum, or acombination thereof; and adjusting one or more of the photosyntheticefficiency parameters to drive the photosynthesis by the delivery of anamount of light to optimize light absorption of the photosyntheticorganism while providing enough dark time between light pulses toprevent oversaturation of the chlorophyll reaction centers.
 2. Themethod of claim 1, wherein the chlorophyll fluorescence feedback controlsystem includes a photosynthetic organism, a fluorometer, and a LEDilluminating system.
 3. The method of claim 1, wherein the pulsed lightis derived from a source that comprises one or more of: a Light-EmittingDiode (LED), Organic Light-Emitting Diode (OLED), or a combinationthereof.
 4. The method of claim 1, wherein the pulse rate is betweenabout 500 Hz to 10 kHz.
 5. The method of claim 1, wherein a wavelengthof the pulsed light is selected from the group consisting of: blue LED(440-490 nm), cyan LED (505 nm), white LED (2700 k-10,000 k), and acombination thereof.
 6. The method of claim 1, further comprising:changing the light intensity of the spectral composition of pulsed lightby decreasing the intensity of wavelengths that are strongly absorbedand increasing the intensity of wavelengths that are weakly absorbed toallow deeper penetration of light energy into a culture or canopy of thephotosynthetic organism.
 7. The method of claim 6, wherein the pulsedlight applies blue (440-490 nm) and red (600-680 nm) light to theculture or canopy and the culture or canopy has strong absorption in theblue (440-490 nm) and red (600-680 nm) regions.
 8. The method of claim7, wherein the culture increases its density during cultivationpreventing effective light penetration into the culture or canopy andinducing increased energy dissipation as heat (NPQ), wherein thebiological optimization system decreases the intensity of blue and redillumination and replaces the intensity of illumination with colorshaving higher reflection, wherein the colors having higher reflectionare selected from the group consisting of: cyan (495-515 nm), green(520-540 nm), orange/amber (565-595 nm), and a combination thereof.
 9. Abiological optimization system for enhancing photosynthetic efficiencyof a photosynthetic organism comprising: a source of pulsed light; and achlorophyll fluorometer.
 10. The biological optimization system of claim9, wherein the photosynthetic organism comprises a plant or animalutilizing chlorophyll as an energy collector/converter.
 11. Thebiological optimization system of claim 10, wherein the photosyntheticorganism is selected from the group consisting of: microalgae,macroalgae, terrestrial plant, coral, corallimorph, anemone, clam, ahost organism containing a photosynthetic symbiotic organism, and acombination thereof.
 12. The biological optimization system of claim 11,wherein the microalgae is selected from the group consisting of:Chlorella sorokiniana, Chlorella minutissima, and a combination thereof.13. The biological optimization system of claim 9, wherein the source ofpulsed light is a LED illuminating system.
 14. The biologicaloptimization system of claim 9, wherein the chlorophyll fluorometerprovides chlorophyll fluorescence feedback to a chlorophyll fluorescencefeedback control system, wherein the chlorophyll fluorescence feedbackcontrol system adjusts the output of the source of pulsed light.
 15. Thebiological optimization system of claim 14, wherein the chlorophyllfluorescence feedback includes one or more of the following:photosynthetic efficiency, photochemical processing (qP), or waste heatdissipation(NPQ or qN), of the photosystem.
 16. The biologicaloptimization system of claim 14, wherein the chlorophyll fluorescencefeedback is utilized adjust one or more of the following: a pulse rate,pulse on/off duration, light intensity, or light spectrum, of the sourceof pulsed light to provide precisely enough light to excite thephotosynthetic organism without oversaturation and photoinhibition withless energy loss through heat dissipation (NPQ).
 17. The biologicaloptimization system of claim 16, wherein the pulse rate is selected fromthe group consisting of: about 500 Hz, about 1,000 Hz, about 2,000 Hz,about 2,500 Hz, about 3,000 Hz, about 3,500 Hz, about 4,500 Hz, about5,000 Hz, about 10,000 Hz, about 20,000 Hz, about 50,000 Hz, and acombination thereof.
 18. The biological optimization system claim 16,wherein one or more of the following: the pulse rate, pulse on/offduration, light intensity, and light spectrum, is adjusted duringillumination based upon the chlorophyll fluorescence feedback data inreal time.
 19. The biological optimization system of claim 9, whereinthe need for non-photochemical quenching by the organism is reducedcompared to continuous illumination.
 20. The biological optimizationsystem of claim 9, wherein the energy required by the source of pulsedlight is decreased due to the duty-cycle of 50% or less.
 21. Thebiological optimization system of claim 9, wherein the light intensityof the spectral composition of light from the illumination changes bydecreasing the intensity of wavelengths that are strongly absorbed andincreasing the intensity of wavelengths that are weakly absorbed toallow deeper penetration of light energy into a culture or canopy of thephotosynthetic organism.
 22. The biological optimization system of claim21, wherein the illumination applies blue (440-490 nm) and red (600-680nm) light to the culture or canopy and the culture or canopy has strongabsorption in the blue (440-490 nm) and red (600-680 nm) regions. 23.The biological optimization system of claim 22, wherein the illuminatedculture increases its density during cultivation preventing effectivelight penetration into the culture or canopy and inducing increasedenergy dissipation as heat (NPQ), wherein the biological optimizationsystem decreases the intensity of blue and red illumination and replacesthe intensity of illumination with colors having higher reflection,wherein the colors having higher reflection are selected from the groupconsisting of: cyan (495-515 nm), green (520-540 nm), orange/amber(565-595 nm), and a combination thereof.